This proposal involves a genetic approach to studying the possible relationships between growth, DNA synthesis, and cellular differentiation of a pluripotent, malignant embryonal carcinoma cell line, PSA-1. One aspect of the proposal involves the study of DNA synthesis at a cellular, chromosomal, and molecular level as the embryonal carcinoma cell line differentiates to form embryoid bodies in vitro. Another aspect of this proposal will be the selection and characterization of temperature sensitive mutants in two embryonal carcinoma cell lines, PSA-1 and LT-1-2A, which will still differentiate at the permissive growth temperature. These mutants will be genetically characterized into complementation groups and biologically characterized for growth at permissive and restrictive temperatures. Combining the information from the DNA synthesis study with the different classes of temperature sensitive mutants obtained should allow pursuance of answers to the following questions: 1) Are conditional stem cell mutations for growth also expressed in the differentiated cell types generated by the stem cell? 2) Can a differentiative transition be inhibited or initiated by controlling growth or DNA synthesis? 3) Can the inactivation of X-chromosome linked genes be controlled with temperature sensitive growth mutants?